Multilocus sequence typing reveals three genetic subpopulations of Cryptococcus neoformans var. grubii (serotype A), including a unique population in Botswana

Abstract

We applied multilocus sequence typing (MLST) to investigate the population structure and mode of reproduction of Cryptococcus neoformans var. grubii (serotype A). This MLST system utilizes 12 unlinked polymorphic loci, which are dispersed on nine different chromosomes, and allows the unambiguous identification of closely related strains of serotype A. We compared MLST analyses with the conventional genotyping method of detecting amplified fragment length polymorphisms (AFLPs), and there was excellent correlation between the MLST and AFLP results. However, MLST differentiated a larger number of strains. We analyzed a global collection of isolates of serotype A using both methods, and the results identified at least three genetically distinct subpopulations, designated groups VNI, VNII, and VNB. Groups VNI and VNII are widespread, dominated by isolates with the MATalpha mating type, and predominantly clonal. Conversely, isolates of group VNB are unique to Botswana, include a significant proportion of fertile strains with the MATa mating type, and manifest compelling evidence of recombination. We have AFLP genotyped >1000 strains of serotype A from different parts of the world, including isolates from several African countries, and, to date, haploid serotype A isolates of group VNB have been found only in Botswana.

Figure 1.

Genetic relationships among the 47 unique AFLP genotypes (A) and the 57 unique MLST genotypes (B) of C. neoformans serotype A visualized by a nonmetric MDS plot. Squares represent unique genotypes from Botswana, and triangles represent unique genotypes from other countries.

Figure 2.

(A) Genetic relationships of MLST genotypes among the 102 isolates of C. neoformans serotype A visualized by the neighbor-joining dendrogram. Numbers on each branch indicate the bootstrap values >50%, based on 500 replications. Vertical lines represent strains with identical genotypes. The clades labeled A1–A5 and A10 are AFLP genotypes that correspond to the AFLP genotypes listed in Table 1. VNB-A and VNB-B are two subpopulations within the VNB group described previously (Litvintseva et al. 2003). Isolates from Botswana are shown in red, and isolates with the MATa mating type are shown in boldface type and designated with “a.” (B) Strict consensus of eight maximum-parsimony trees inferred from the combined gene genealogies of the C. neoformans serotype A MLST genotypes and rooted with sequences of the JEC21 serotype D strain (Loftus et al. 2005). Only unique genotypes are included. However, large gaps in the alignments of nucleotide sequences from strains of serotype A and the serotype D strain reduced the number of unique genotypes from 57 to 27. Ten strains that were inconsistently placed within the gene genealogies of the 12 genes were excluded from the analysis. CI, consistency index; RI, retention index.

Figure 2.

(A) Genetic relationships of MLST genotypes among the 102 isolates of C. neoformans serotype A visualized by the neighbor-joining dendrogram. Numbers on each branch indicate the bootstrap values >50%, based on 500 replications. Vertical lines represent strains with identical genotypes. The clades labeled A1–A5 and A10 are AFLP genotypes that correspond to the AFLP genotypes listed in Table 1. VNB-A and VNB-B are two subpopulations within the VNB group described previously (Litvintseva et al. 2003). Isolates from Botswana are shown in red, and isolates with the MATa mating type are shown in boldface type and designated with “a.” (B) Strict consensus of eight maximum-parsimony trees inferred from the combined gene genealogies of the C. neoformans serotype A MLST genotypes and rooted with sequences of the JEC21 serotype D strain (Loftus et al. 2005). Only unique genotypes are included. However, large gaps in the alignments of nucleotide sequences from strains of serotype A and the serotype D strain reduced the number of unique genotypes from 57 to 27. Ten strains that were inconsistently placed within the gene genealogies of the 12 genes were excluded from the analysis. CI, consistency index; RI, retention index.

Figure 3.

Most parsimonious trees for 102 isolates of C. neoformans serotype A based on sequence polymorphisms in the following genes: CAP10 (A), SOD1(B), and MP88 (C) rooted with sequences of the JEC21 serotype D strain. For clarity of presentation only selected isolates from Botswana are shown. Vertical lines represent identical genotypes. Numbers above each branch indicate bootstrap values >50%, based on 500 replications. Strains that are inconsistently placed within the gene genealogies are shown in boldface type and marked with arrows. CI, consistency index; RI, retention index.